Researchers in the biological sciences are greatly interested in obtaining dynamic chemical information about the processes that occur in living systems. Dynamic chemical information relates to attempts to obtain either continuous, or near-continuous samples from the living organism. By obtaining a large number of closely temporally spaced samples of the material of interest from the living organism, one can better obtain information about the interaction between the living organism and the material of interest over a selected time interval.
The use of continuous sampling can permit the user to better elucidate the pathways and kinetics of absorption of a particular compound of interest, to determine how the compound is transformed within the body, and to determine the rate at which the compound is eliminated from the living organism. This information is often necessary to permit the researcher to fully assess the safety of both pharmaceutical and environmental compounds. For example, one might desire information about the reaction of an analgesic compound in a living organism. To determine this, one could give a known amount of the compound to the organism, and obtain continuous tissue samples (e.g., brain fluid) from the animal. By analyzing the brain fluid samples that were withdrawn at various time intervals after giving the analgesic to the animal, one could obtain a great deal of information about the manner in which the analgesic functioned within the living organism. For example, one could determine the amount of time required by a living organism to absorb the analgesic, and to transfer the analgesic to the site (e.g. the brain) from which the sample was being drawn. Further, one could construct suitable experiments to determine the quantity of analgesic that actually arrived at the site of interest, and also determine whether the particular analgesic is being transformed by the body into break-down products, or other compounds different than the analgesic compound itself.
Also, by continuously obtaining samples, one can also determine the amount of time required to remove the particular analgesic system from the living organism. Knowing the rate of elimination of the analgesic can be especially important for enabling its manufacturer to either adjust the dosage, or alter the drug (such as through micro encapsulation) to enable the drug to achieve a proper liter within the living organism for a proper time period.
When determining pharmacokinetic parameters for a particular compound of interest, or its metabolites, it is often necessary to determine these parameters at specific tissue sites. Other times, it is necessary to determine these parameters in the general system of the living organism. The time scale in which the reactions of interest take place, and in which samples must be removed are typically on the order of minutes to days after the compound is given to the organism.
One goal when performing continuous or semi-continuous sampling is to improve the temporal resolution of the sampling and detection process. Temporal resolution relates to the number of data points that one can obtain over a given time span. Figures for temporal resolution are typically given in units of time span. For example, to say that a particular experiment had a temporal resolution of 1 minute means that a data point was taken (or capable of being taken) each minute. Typically, the factor limiting temporal resolution is the time required to accumulate and collect a sample of a size adequate for the particular detection method being used in conjunction with the sampling. As will be appreciated, a lower sample volume requirement will generally enable a better temporal resolution to be achieved.
A good temporal resolution is especially important when detecting the presence of transient compounds that may remain at a tissue site for only a very short time duration. For example, neuropharmacological investigations, such as investigation into the release of neurotransmitters in response to amphetamine or cocaine, require the acquisition of chemical information from specific brain regions, with temporal resolution of seconds to minutes. Stated another way, because the volume and presence of particular compounds are likely to change so rapidly within such a short time period, a large number of closely temporally spaced data points are necessary to obtain truly meaningful kinetic information.
Current methods for obtaining time interval type, sequential multiple sample chemical information from tissues of living systems have usually involved either a postmortem analysis taken at several time points, or the use of biosensors implanted in vivo. Postmortem analyses typically require the use of a large number of animals, with only a single sample being taken from each animal. The "pseudo continuity" of samples is achieved by taking the samples from the different animals at different points in time. The use of postmortem analyses to construct a temporal plot of chemical events is difficult, and often provides ambiguous results. While several compounds can be determined at each time point (and hence from each sample) each animal can provide data for only a single time point. It is therefore preferable in terms of both the quality of data obtained, and from the standpoint of reducing the number of experimental animals needed, to obtain the entire time course (data set) from one animal, by taking a large number of samples over a span of time from the single animal. Although this can be accomplished through the use of a biosensor, biosensors suffer the drawback of providing less chemical information since they are usually limited to monitoring only a single chemical species.
One other difficulty that inhibits the removal of a large number of samples from an animal over a course of time is the generally small volume of sample available. This is especially true when the living organism is a small laboratory animal, such as a rat.
To overcome the sampling difficulties, many researchers use a microdialysis sampling technique. Microdialysis sampling is accomplished by implanting a microdialysis probe that consists of a small, semi-permeable membrane fiber at the site of interest. This fiber is slowly perfused with a sampling solution. The semipermeable microdialysis membrane allows certain molecules of interest to pass from the animal into the sampling solution. Small molecules in the extra-cellular space can diffuse into the microdialysis membrane fiber, and are swept away by the sampling solution, to be collected for analysis. Microdialysis probes can be implanted in many tissues with minimal discomfort to the experimental animal.
The introduction of microdialysis sampling has provided a technique that can continuously monitor chemical reactions in vivo. By using the appropriate analytical method, several compounds can be determined simultaneously. Therefore, microdialysis can provide both the temporal and chemical information needed to determine the behavior and characteristics of a compound of interest over a relatively long time span. As such, microdialysis can provide the information necessary to permit the researcher to fully elucidate certain biochemical processes.
However, one limitation of microdialysis has been the limited temporal resolution that has been achievable to date. While microdialysis is a continuous sampling technique, it is typically coupled to a separation method that requires discrete samples. For example, with liquid chromatography, a discrete "plug" of a sample containing several compounds is inserted at one time into the upstream end of the liquid chromatography column. As the sample flows downstream through the liquid chromatography column, the several compounds in the sample "plug" are separated into discrete bands of individual compounds, which can then be detected. If a several-compound-containing sample were injected as a continuous flow into a liquid chromatography column, no discrete bands would emerge. Rather, the output would be a mixture not unlike the sample that was inserted into the upstream end of the liquid chromatography column.
The temporal resolution of an experiment wherein the sample is withdrawn from a living organism through a microdialysis technique is determined by a combination of the perfusion rate of the compound of interest through the microdialysis probe, and the sample volume requirement of the particular analytical technique. As such, the greater the volume of analyte required for the particular analytical technique, the slower the temporal resolution.
By necessity, microdialysis samples are aqueous. The analytes sought to be detected typically have a small molecular weight, and a moderate to high water solubility. For these reasons, liquid chromatography has been the most popular analytical technique to couple with microdialysis sampling for separating out the various compounds of interest within the microdialysis sample. However, even with the use of very small diameter (e.g. 1 mm) microbore columns, most liquid chromatography techniques require at least one microliter of sample to perform the necessary separation and detection operations.
Typically, microdialysis sampling uses a very slow perfusion rate, such a 1 microliter per minute. At such a rate, the maximum temporal resolution when using a liquid chromatograph requiring 1 micro liter of sample is only 1 detectable sample per minute. Theoretically, higher perfusion rates can be used to generate larger sample volumes per unit of time, and thus theoretically reduce the maximum temporal resolution. However, higher perfusion rates may often not reduce the effective temporal resolution, as higher perfusion rates usually result in the sample having a lower concentration of the analyte of interest, thereby placing a strain on the detection limits of the analytical method.
Certain other methods exist that require smaller sample volumes than liquid chromatography. One such analytical method is capillary electrophoresis, in which sample volumes of a few nanoliters are usually sufficient. Because of its smaller volume requirements, the use of capillary electrophoresis with microdialysis can provide improved temporal resolution, when compared to liquid chromatography.
It is known that microdialysis samples can be analyzed by capillary electrophoresis. See, O'Shea, Telting-Diaz, Lunte and Lunte Electroanaly. 1992, Volume 4 at Pages 463, 468; O'Shea, Weber, Bammel, Lunte, Lunte and Smith, Chromatographer, 1992, Volume 608 at Pages 189-195; and Tellez, Forges, Roussin, and Hernandez, J. Chromatogr. Biomed. Appl., 1992, Volume 581 at Pages 257-266. These known earlier studies used an off-line collection of the microdialysis sample, and a subsequent analysis by capillary electrophoresis. Typically, five microliters of the dialysate was collected for analysis. However, of the five microliters collected, only five nanoliters were ultimately injected into the capillary electrophoresis detection system. The reason that a quantity greater than five nanoliters was collected is due to the difficulties involved in quantitatively handling volumes less than one microliter in off-line systems. In particular, evaporation creates significant problems.
One advantage with the use of microdialysis sampling is that the dialysate (the sampling material and the fluid that passes through the microdialysis membrane) is protein-free and suitable for direct injection into either a liquid chromatography column or into a capillary electrophoresis system. By coupling the microdialysis sampling to an analytical separator/detector in an on-line fashion, it may be possible to avoid the problems of sample manipulation discussed above. To this end, on-line microdialysis sampling has been described as being coupled to liquid chromatography, flow injection analysis, and mass spectrometry. See Damsma, Westernik, DeVreis, Van den Berg, and Horn, J. Neurochem 1987, Volume 48 at Pages 15-23--15-28 (liquid chromatography); Church and Justice Anal. Chem., 1987, Volume 59 at Pages 7-12 through 7-16 (liquid chromatography); Boutelle, Fellows, and Cook, Anal. Chem., 1992, Volume 64 at Page 1790 through 1794 (flow injection analysis); and Caprioli and Lyn, Proc. Natl. Acad. Sci. U.S.A., 1990, at Pages 240-243 (mass spectrometry).
With the on-line systems discussed above, to the applicant's knowledge, the best temporal resolution achieved by any of the techniques has been two minutes.
Capillary electrophoresis systems have also been coupled to liquid chromatography in a multi-dimensional separation system, and to a flow injection analysis (FIA) system. See Bushey and Jorgenson, Anal. Chem., 1990, Volume 62 at Pages 978-984 (multi-dimensional separation system); Jorgenson and Bushey U.S. Pat. No. 5,131,998; and Tsuda and Zare, J. Chromatogr., 1991, Volume 559 at Pages 103-110. However, none of the systems described above is suitable to couple a microdialysis sampling system to a capillary electrophoresis detection system. It is therefore one object of the present invention to provide a means for coupling a capillary electrophoresis detection system to a sequential, multiple sample acquisition device such as a microdialysis or ultrafiltration sampling system.